The present invention relates to a method for the diagnosis of Alzheimer""s dementia (AD). The invention particularly relates to a process for quantifying the presence of at least one biochemical marker associated with Alzheimer""s dementia. More particularly, the invention relates to a point-of-care immunoassay which utilizes unique antibodies to enable the differential diagnosis of Alzheimer""s versus non-Alzheimer""s forms of dementia.
Alzheimer""s disease, also referred to as Alzheimer""s dementia or AD is a progressive neurodegenerative disorder that causes memory loss and serious mental deterioration. Diagnosticians have long sought a means to definitively identify AD during the lifetime of demented patients, as opposed to histopathological examination of brain tissue, which is the only present means available for rendering an ultimate diagnosis of AD. AD is the most common form of dementia, accounting for more than half of all dementias and affecting as many as 4 million Americans and nearly 15 million people worldwide. Dementia may start with slight memory loss and confusion, but advances with time reaching severe impairment of intellectual and social abilities. At age 65, the community prevalence of AD is between 1-2%. By age 75, the figure rises to 7%, and by age 85 it is 18%. The prevalence of dementia in all individuals over age 65 is 8%. Of those residing in institutions, the prevalence is about 50%, at any age.
The social impact of this disease is enormous, caused by the burden placed on caregivers, particularly in the latter stages of the disease. The substantial economic costs are largely related to supportive care and institutional admission. The rapidly increasing proportion of elderly people in society means that the number of individuals affected with AD will grow dramatically, therefore finding an early accurate diagnosis and a cure for AD is becoming an issue of major importance world wide.
When an individual is suspected of AD, several recommended tests are performed: (1) Mini Mental State Examination (MMSE)xe2x80x94an office-based psychometric test in the form of a Functional Assessment Questionnaire (FAQ) to examine the scale for functional autonomy, (2) Laboratory testsxe2x80x94complete blood count, measurement of thyroid stimulating hormone, serum electrolytes, serum calcium and glucose levels, (3) Neuroimagingxe2x80x94most commonly used is computed tomography (CT) which has a role in detecting certain causes of dementia such as vascular dementia (VaD), tumor, normal pressure hydrocephalus or subdural hematoma. However, neuroimaging is less effective in distinguishing AD or other cortical dementias from normal aging. In primary care settings, some suggest that CT could be limited to atypical cases, but others recommend routine scanning. Magnetic resonance imaging (MRI) currently offers no advantage over CT in most cases of dementia.
While Alzheimer""s is the most common form of dementia, accounting for at least 60% of cases, diagnostic procedures for determining the exact cause of dementia, among more than 80 different species, is difficult at best. Furthermore, the currently performed tests are inadequate in differentiating AD from other types of dementia.
In comparison to other disease areas, the field of dementia raises questions concerning the value of diagnosis, since there is currently no cure or effective therapy available. In dementia, as in all other branches of medicine, the certainty of a diagnosis has an important impact on the management of the patient. While AD cannot be cured at present time, there is symptomatic treatment available and the first drugs (acetylcholinesterase for the temporary improvement of cognition and behavior are now licensed by the U.S. Food and Drug Administration. Other drugs are at different stages of clinical trials: (1) Drugs to prevent decline in AD-DESFERRIOXAMINE, ALCAR, anti-inflammatory drugs, antioxidants, estrogen, (2) Neurotrophic Factors: NGF, (3) Vaccine: the recent most exciting report by Schenk et al. (Nature 1999;400:173-7) raises the hope of a vaccine for AD.
The specificity of the various therapies thus require sophisticated diagnostic methodologies, having a high degree of sensitivity for AD, in order to insure their success.
Currently there are a multitude of tests available which aid in the diagnosis of AD. However, the only true existing diagnosis is made by pathologic examination of postmortem brain tissue in conjunction with a clinical history of dementia. This diagnosis is based on the presence in brain tissue of neurofibrillary tangles and of neuritic (senile) plaques, which have been correlated with clinical dementia. Neuritic plaques are made up of a normally harmless protein called amyloid-beta. Before neurons begin to die and symptoms develop, plaque deposits form between neurons early on in the disease process. The neurofibrillary tangles are interneuronal aggregates composed of normal and paired helical filaments and presumably consist of several different protein The internal support structure for brain neurons depends on the normal functioning of a protein called tau. In Alzheimer""s disease, threads of tau protein undergo alterations that cause them to become twisted. The neurohistopathologic identification and counting of neuritic plaques and neurofibrillary tangles requires staining and microscopic examination of several brain sections. However, the results of this methodology can widely vary and is time-consuming and labor-intensive.
Given the ability of both current and prospective pharmacological therapies to forestall and/or reverse the onset and/or progress of Alzheimer""s dementia, an early diagnosis of AD will assist to better manage the care of patients. There are many cases where non-AD dementia could be confused with AD dementia. Such examples include small, undetected strokes which temporarily interrupt blood flow to the brain. Clinically depressed patients or those with Parkinson""s disease can also experience lapses in memory. Many older people are on a variety of medications which as a side effect may, alone or in conjunction, impair their ability to perform cognitive tasks.
Thus, if diagnostic techniques for the early differentiation of AD could be provided, physician""s would achieve an enhanced ability to prescribe appropriate intervention at an early stage in the pathogenesis of this disease.
Various biochemical markers for AD are known and analytical techniques for the determination of such markers have been described in the art. As used herein the term xe2x80x9cmarkerxe2x80x9d xe2x80x9cbiochemical markerxe2x80x9d or xe2x80x9cmarker proteinxe2x80x9d refers to any enzyme, protein, polypeptide, peptide, isomeric form thereof, immunologically detectable fragments thereof, or other molecule that is released from the brain during the course of AD pathogenesis. Such markers include, but are not limited to, any unique proteins or isoforms thereof that are particularly associated with the brain.
Glutamine synthetase (GS) is recognized as an astrocyte-specific enzyme involved in the regulation of ammonia and glutamate metabolism that is over-expressed following brain injury (Norenberg and Martinex-Hernandez, Brain Res 1979;161:303). A few studies on the clinical role of glutamine synthetase have been reported: Gunnersen and Haley (Proc Natl Acad Sci USA 1992;89:11949) found monomeric GS protein in 38 of 39 AD cerebrospinal fluid (CSF) samples, Tumani et al. (Arch Neurol 1999;56(10):1241) describe that the concentration of GS in lumbar CSF of patients with AD is increased significantly but nonspecifically (i.e. also increased in VaD, schizophrenia and ALS). On p.1244, the left-hand column, Tumani states that GS was not found in serum.
Neuron-specific gamma-enolase (NSExcex3xcex3) and S100B proteins, abundant in the brain, are also useful markers for assessing the extent of brain damage: NSExcex3xcex3 for neuronal damage and S100B for astrocyte damage. Concentrations of NSE and S100B proteins from cerebrocortical regions have been examined by means of enzyme linked immunosorbent assay (ELISA). The levels of these proteins in frontal cortex of AD patients were found to be significantly elevated (Kato et al. J Mol Neurosci, 1991;3(2):95). Activated astrocytes over-expressing S100B have been intimately associated with the neuritic xcex2-amyloid plaques of AD (Sheng et al. J Neurosci Res, 1994;39:398, Mrak et al. J Neuropathol Exp Neurol 1996;55:273).
There are a number of different potential uses for biomarkers in AD evaluation, and each use could involve a different marker or set of markers. Such uses may include, but are not limited to, the use of a marker to distinguish AD from other causes of dementia; distinguishing dementia from the non-pathological effects of aging; monitoring the progress of the disease after clinical symptoms become apparent; utilization of a surrogate to monitor the efficacy of the forthcoming therapies for AD; and isolating markers which have utility as risk assessment factors for AD; and identifying both the earliest biological changes occurring in the brain and other changes that occur as the disease progresses. Ideally, it would be preferable to isolate a single marker to fulfill all requirements with a high degree of sensitivity and specificity, however this may be an unreasonable goal. Any individual marker needs to be assessed by sensitivity, specificity, reliability and validity for the type of clinical situation to which it is meant to apply. A marker which is poor at distinguishing AD from other causes of dementia, could nevertheless be an excellent marker for monitoring the progression of the disease process or the response to therapy.
With regard to diagnostic devices, the clinical evaluation and use of point-of-care tests utilizing biological markers are valuable tools for evaluating risk, monitoring disease progression and guiding therapeutic interventions. The advantages which flow from the use of biological markers as diagnostic tools include strengthening the certainty of the clinical diagnosis, distinguishing AD from other causes of dementia, and quantifying the severity of the disease and rate of progression. In addition, tests using biological markers should be rapid, non-invasive, simple to perform and inexpensive.
What is lacking in the art is a relatively non-invasive method and device therefore effective for definitively diagnosing Alzheimer""s dementia in living patients. Additionally, a definitive method of assessing the risk of developing AD is greatly needed.
U.S. Pat. No. 5,445,937 to Haley teaches a method for the diagnosis of Alzheimer""s disease, as well as a means for the diagnosis and differentiation of other diseases. This is done by use of a disease-specific biochemical marker, glutamine synthetase (GS) and its respective photoaffinity label, or labeled antibody specific for GS, at GS""s binding site. The ""937 patent focuses on examining cerebrospinal fluid (CSF) to detect the presence of a photoaffinity labeled, or labeled antibody, nucleotide binding protein and correlates the subsequent level with the presence of AD. Haley teaches a variety of immunoassay techniques to accomplish such a method. While Haley hypothesizes regarding the prophetical utility of diagnostic methods which use blood as a sample and further suggests that a monoclonal and/or polyclonal antibody immunoassay could be developed, he nevertheless fails to reduce either to practice. Thus, the ""937 patent is only useful in teaching a diagnostic test which utilizes cerebrospinal fluid. Obtaining a sample of cerebrospinal fluid entails invasive techniques quite uncomfortable to a patient and requires a lengthy period of time to accomplish. In addition, the only polyclonal and/or monoclonal antibodies suggested by Haley are those having a specificity to sheep brain GS as opposed to a human recombinant form of GS as instantly disclosed herein.
In U.S. Pat. No. 5,508,167, Roses et al. describe methods for diagnosing AD involving the detection of an apolipoprotein E type 4 (ApoE4) isoform or DNA encoding ApoE4. The methods can use blood samples and are analyzed by an immunochemical assay. The blood sample is optionally combined with a reducing agent to reduce the disulfide bond in cysteine residues to the corresponding reactive sulfhydryl groups. Roses et al. further describes a kit for detection of the ApoE4 isoform. The test is based on the differences in the amino acid sequences of the three major ApoE isoforms. The test is not specific for human GS nor does it have sensitivity in differentially diagnosing AD versus non-AD dementia.
Tumani et al. (Arch. Neurol., (1999) 56, pp1241-1246) examine the levels of GS in CSF and the examination of serum in order to determine whether GS is a useful biochemical marker in the diagnosis of AD. Analysis is by an ELISA utilizing a biotin-labeled monoclonal antibody directed against sheep brain GS. Normal ranges of GS concentration are reported as 4 pg/mL in human CSF and 36 pg/mL in human serum. The samples of CSF in AD patients are elevated with a mean level of GS concentration of 20xc2x112 pg/mL, with ALS patients at 13xc2x113 pg/mL, and vascular dementia (VaD) patients at a mean elevated level 13xc2x17 pg/mL. Vascular dementia and ALS patients show a slightly lower increase. Patients with AD are measured with mean levels of 111xc2x153 pg/mL in serum. However, patients with amyotrophic lateral sclerosis (ALS) and vascular dementia also present with mean elevated levels at 116xc2x162 pg/mL and 72xc2x159 pg/mL in serum, respectively. Thus, no definitive diagnosis regarding AD dementia or differential diagnosis between AD versus non-AD dementia could be elucidated from these assays.
Gunnersen and Haley (Proc. Natl. Acad. Sci. (1992) 89, pp11949-11953) provides evidence of GS detected in CSF of patients with AD but not in that of healthy control subjects or controls with other diseases. The other diseases under consideration are epilepsy, ALS, and Parkinson""s. Patients with ALS or Pick""s disease in addition to AD do show positive results, meanwhile ALS patients do not show positive results, indicating GS is specific to AD. As with other publications, antibodies raised against non-human GS are utilized for detected of GS.
Generally, most scientific papers tend to focus on the peptide, xcex2-amyloid, since it is postulated to be a major determinant of AD. This is supported by the observation that certain forms of familial AD mutations result in the over production of xcex2-amyloid, particularly the longer form (1-42) which aggregates more readily than the shorter form. Hensley et al. (Proc. Natl. Acad. Sci., (1994), 91, pp3270-3274) examine the neurotoxicity based on free radical generation by the peptide xcex2-amyloid in its aggregation state. Several synthetic fragments of the peptide are tested for resulting neurotoxicity. Based on the fact that oxygen seems to be a requirement for radical generation and glutamate synthetase and creatine kinase enzymes are oxidation-sensitive biomarkers, the inactivation of these enzymes are utilized as indicators of active attack on biological molecules by these fragmented xcex2-amyloid aggregates.
The present invention relates to a method for the diagnosis of Alzheimer""s dementia (AD), particularly to a method for diagnosing and differentiating Alzheimer""s dementia from other forms of dementia by testing for the presence of specific biochemical markers for Alzheimer""s disease in bodily fluids, particularly in blood, blood products, urine, saliva and the like. The invention further relates to a process for quantifying the presence of at least one biochemical marker associated with Alzheimer""s dementia. More particularly, the invention relates to a point-of-care immunoassay which utilizes unique antibodies to enable the differential diagnosis of Alzheimer""s versus non-Alzheimer""s forms of dementia.
The present invention relates to methods and ELISA system for diagnosing, subtyping and monitoring Alzheimer""s disease. The invention is based on the discovery that S100B, NSExcex3xcex3 and GS proteins are released from the brain and can be detected in body fluids outside the brain.
The generation and purification of recombinant human GS are described. These GS proteins may be used to generate monoclonal or polyclonal antibodies that, in turn, can be used in immunoassays, wherein they enter into an immunoreaction which can be monitored and/or quantified to detect circulating GS protein in suspected individuals. Alternatively, the GS protein themselves may be used in immunoassays to detect circulating autoantibodies in such individuals. The occurrence of Alzheimer""s dementia is characterized by the recognition of levels of a particular biochemical marker in bodily fluid, said levels correlating to the manifestation of Alzheimer""s dementia symptoms as quantified by MMSE testing. As a risk assessment test, the recognition of levels of such markers which are indicative of the development of Alzheimer""s dementia further augments the diagnostic capability afforded to the skilled practitioner.
Accordingly, it is an objective of the instant invention to provide a relatively non-invasive and highly sensitive method for the definitive diagnosis of Alzheimer""s disease.
It is a further objective of the invention to provide a method which includes analysis of at least one body fluid of a patient to determine the presence of at least one marker indicative of AD vs non-AD dementia.
It is a further objective of the instant invention to provide antibodies specific to neuronal related proteins, as identified by the method of the current invention.
It is a still further objective of the instant invention to provide an immunoassay effective for the recognition of neuronal specific proteins in one or more human bodily fluids.
It is yet another objective of the instant invention to provide a purified monoclonal antibody specific for human glutamine synthetase.
It is a still further objective of the invention to provide a test kit for the diagnosis of AD comprising a non-invansive point-of-care test which can be performed utilizing a sample comprising blood or any blood product.